The process of deliberately creating changes in the genetic material of living cells has the goal of modifying one or more genetically encoded biological properties of that cell, or of the organism of which the cell forms part or into which it can regenerate. These changes can take the form of deletion of parts of the genetic material, addition of exogenous genetic material, or changes in the existing nucleotide sequence of the genetic material. Methods of altering the genetic material of eukaryotic organisms have been known for over 20 years, and have found widespread application in plant, human and animal cells and micro-organisms for improvements in the fields of agriculture, human health, food quality and environmental protection. The most common methods consist of adding exogenous DNA fragments to the genome of a cell, which will then confer a new property to that cell or its organism over and above the properties encoded by already existing genes (including applications in which the expression of existing genes will thereby be suppressed). Although many such examples are effective in obtaining the desired properties, these methods are nevertheless not very precise, because there is no control over the genomic positions in which the exogenous DNA fragments are inserted (and hence over the ultimate levels of expression), and because the desired effect will have to manifest itself over the natural properties encoded by the original and well-balanced genome. On the contrary, methods of targeted mutagenesis that will result in the addition, deletion or conversion of nucleotides in predefined genomic loci will allow the precise modification of existing genes. In addition, due to the precise nature of targeted mutagenesis, its is expected that novel plant lines obtained in this way will be more readily accepted by consumers.
Targeted mutagenesis is a site-directed mutagenesis method that is based on the delivery into the eukaryotic cell nucleus of synthetic mutagenic nucleobases (molecules consisting of short stretches of nucleotide-like moieties that resemble DNA in their Watson-Crick basepairing properties, but may be chemically different from DNA) (Alexeev and Yoon, Nature Biotechnol. 16: 1343, 1998; Rice, Nature Biotechnol. 19: 321, 2001; Kmiec, J. Clin. Invest. 112:632, 2003). Once introduced into the cell, such mutagenic nucleobases basepair with the complementary sequence at the target locus. By deliberately designing a mismatch in the nucleobase, the mismatch may a nucleotide conversion at the corresponding position in the target genomic sequence. This method allows the conversion of single or at most a few nucleotides in endogenous loci, but may be applied to create stop codons in existing loci, resulting in a disruption of their function, or to create codon changes, resulting in genes encoding proteins with altered amino acid composition (protein engineering).
Targeted mutagenesis has been described in plant, animal and yeast cells. Two different classes of synthetic mutagenic nucleobases have been used in these studies, the chimeric DNA:RNA nucleobases or single stranded nucleobases.
The chimeric DNA:RNA nucleobases (chimeras) are self complementary molecules consisting of a 25 bp DNA only region and a 25 bp complementary sequence made up of 5 bp of core region of DNA flanked on either side by 10 bp of 2′-O-methylated RNA that are thought to aid stability of the chimera in the cell. The 5 bp core region includes in its centre an engineered mismatch with the nucleotide to be altered in the genomic target DNA sequence. Both these regions are linked by 4 bp thymidine hairpins. Upon introduction into the cell the chimera is thought form a double D-loop with its target sequence and a mismatch is formed between the chimera and the target nucleotide. This mismatch is then resolved by endogenous cellular DNA repair proteins by conversion of the genomic nucleotide. The first examples of targeted mutagenesis using chimeras came from animal cells (reviewed in Igoucheva et al. 2001 Gene Therapy 8, 391-399) and were then also later used to achieve targeted mutagenesis in plant cells (Beetham et al. 1999 Proc. Natl. Acad. Sci. USA 96: 8774-8778; Zhu et al. 1999 Proc. Natl. Acad. Sci. USA 96, 8768-8773; Zhu et al. 2000 Nature Biotech. 18, 555-558; Kochevenko et al. 2003 Plant Phys. 132: 174-184; Okuzaki et al. 2004 Plant Cell Rep. 22: 509-512). Unlike human cells, a plant cell in which a targeted mutagenesis event has occurred can be regenerated into an intact plant and the mutation transferred to the next generation, making it an ideal tool for both research and commercial mutagenesis of important food crops. However, extensive research by many laboratories has shown that the targeted mutagenesis frequency using chimeras is quite low and variable, or not even detectable (Ruiter et al. 2003 Plant Mol. Biol. 53, 715-729, Van der Steege et al. (2001) Nature Biotech. 19: 305-306), and depended on such factors as the transcriptional status of the target, the position of the cell in the cell cycle, the sequence of the target and the quality of the chimeras, which are difficult to synthesize. Due to the relatively low frequency of targeted mutagenesis with the methods known in the art, such events can only be detected when alteration of a single nucleotide of the genomic target results in a dominant selectable phenotype. In plant cells specific point mutations were introduced into the open reading frame of the acetolactate synthase (ALS, in maize AHAS) gene which catalyzes the initial step common to the synthesis of the branched chain amino acids leucine, isoleucine and valine. In tobacco, single nucleotide alterations are sufficient to produce the codon conversions P194Q or W571L. The ALS protein produced after either of these codon conversions is insensitive to inhibition by the sulfonylurea class of herbicides, thus providing a method of selection for single nucleotide conversions at a chromosomal locus.
Due to the difficulties of working with chimeras, more reliable alternative oligonucleotide designs have been sought. Several laboratories have investigated the ability of single stranded (ss) nucleobases to perform targeted mutagenesis. These have been found to give more reproducible results, be simpler to synthesize, and can also include modified nucleotides to improve the performance of the mutagenic nucleobase in the cell (Liu et al. 2002 Nucl. Acids Res. 30: 2742-2750; review, Parekh-Olmedo et al. 2005 Gene Therapy 12: 639-646; Dong et al. 2006 Plant Cell Rep. 25: 457-65; De Piédoue et al. 2007 Oligonucleotides 17: 258-263).
Targeted mutagenesis has been described in a variety of patent applications of Kmiec, inter alia in WO0173002, WO03/027265, WO01/87914, WO99/58702, WO97/48714, WO02/10364. In WO 01/73002 it is contemplated that the low efficiency of gene alteration obtained using unmodified nucleobases is largely believed to be the result of their degradation by nucleases present in the reaction mixture or the target cell. To remedy this problem, it is proposed to incorporate modified nucleotides that render the resulting nucleobases resistant against nucleases. Typical examples of such modified nucleotides include phosphorothioate linkages or 2′-O-methyl-analogs. These modifications are preferably located at the ends of the nucleobase, leaving a central unmodified domain surrounding the targeted base. In support of this, patent application WO 02/26967 shows that certain modified nucleotides increasing the intracellular lifetime of the nucleobase enhance the efficiency of targeted mutagenesis in an in vitro test system and also at a mammalian chromosomal target. Not only the nuclease resistance, but also the binding affinity of an ss mutagenic nucleobase to its complementary target DNA has the potential to enhance the frequency of targeted mutagenesis dramatically. A ss nucleobase containing modified nucleotides that enhance its binding affinity may more efficiently find its complementary target in a complex genome and/or remain bound to its target for longer and be less likely to be removed by proteins regulating DNA transcription and replication. An in vitro targeted mutagenesis assay has been used to test many modified nucleotides to improve the efficiency of the mutagenesis process. Locked nucleic acids (LNA) and C5-propyne pyrimidines have modifications of the sugar moiety and base respectively that stabilize duplex formation and raise the melting temperature of the duplex. When these modified nucleotides are incorporated on an ss nucleobase, they enhance the efficiency of targeted mutagenesis up to 13 fold above that obtained using an unmodified nucleobase of the same sequence. See in his respect WO2007073166 and WO2007073170 in the name of the present applicants.
The present inventors have set out to improve the frequency of targeted mutagenesis in plant cells by optimizing the method used to introduce the mutagenic nucleobases into plant cells. The most widely used method for transformation of plant cells, Agrobacterium mediated transformation, transfers a section of its tumour inducing (Ti) plasmid, the so-called T-DNA, to plant cells where it efficiently integrates into the plant genome at a random position. The T-DNA is flanked at either end by “border” sequences of up to 22 bps derived from the Ti plasmid which share no homology with the target sequence. Given the short length of the ss mutagenic nucleobases used for targeted mutagenesis, the border sequences would interfere with the process. Thus, targeted mutagenesis can only be achieved in plant cells through direct DNA transfer using chemical or physical methods.
In the literature, several such direct DNA transfer techniques have been reported and include electroporation, polyethylene glycol (PEG) treatment of protoplasts, biolistic bombardment of plant callus material and microinjection of DNA into individual protoplasts or tissue. The art provides no indication as regards a preferred method for the transfer of ss nucleobases for targeted mutagenesis, in particular for DNA transfer to plants or plant protoplasts.
In order to achieve as high a targeted mutagenesis efficiency in plants as possible, the present inventors in the course of their investigations have identified four factors that are to be optimized. First, the mutagenic nucleobase is preferably introduced with a high transformation efficiency, i.e. introduced into as many plant cells as possible. Second, the treatment is preferably not lethal to most of the cells, ensuring that as many cells as possible that are transformed also survive the transformation procedure (survival efficiency). Thirdly, the transformation method is preferably not detrimental to the subsequent divisions of the transformed plant cells to form microcalli (regeneration/plating efficiency) and finally it is preferably possible to identify individual regenerated plants derived from targeted mutagenesis events without application of a selection (identification efficiency).
Most methods for transformation of DNA to individual plant cells use protoplasts, derived directly from leaves (mesophyll protoplasts) or from cell suspensions (reviewed in Sheen, J. (2001) Plant Phys. 127: 1466-1475). Protoplasts can be used for transient expression studies, in which case gene expression or protein localization can be assessed shortly after transformation, or for production of stably transformed plants when the protoplasts are grown on medium to promote callus formation and organogenesis.
Transformation of plant protoplasts using electroporation has been previously reported (Fromm et al. (1985) Proc. Natl. Acad. Sci. USA 82: 5824-5828; Nishiguchi et al. (1986) Plant Cell Rep. 5: 57-60; Ou-Lee et al. (1986) Proc. Natl. Acad. Sci. USA 83: 6815-6819; Hauptmann et al. (1987) Plant Cell Rep. 6: 265-270; Jones et al. (1989) Plant Mol. Biol. 13: 503-511). Generally, the field strength (V/cm) giving the highest transformation efficiency results in <50% of protoplast survival (Jones et al. (1989) Plant Mol. Biol. 13: 503-511). In tobacco electroporation studies we have found that only approximately 10% of the total tobacco protoplasts in the sample are transformed with a plasmid expressing GFP and this relatively low transformation efficiency has also been observed after electroporation of Arabidopsis protoplasts (Miao et al. (2007) Nature Protocols 10: 2348-2353). In general, the optimal electroporation conditions must be determined empirically for each plant species and these can also vary according to the type of electroporation machine and the method and buffers used for protoplast isolation. While electroporation has been successfully applied to many plant species, it remains a difficult technique with several serious limitations (as discussed in: http://genetics.mgh.harvard.edu/sheenweb/faq.html), in particular in terms of reproducibility. Hence electroporation is less desirable for enhancing the overall efficiency for TNE of targeted mutagenesis.
Direct gene transfer using biolistic delivery has been very successful in generating transgenic crop plants and is routinely used for the stable integration of transgenes. Cell suspensions are transferred to solid medium for callus induction and this material is bombarded by gold particles driven by a high pressure gas source. It has been reported that the transformation frequencies are low, ˜0.01% of the total cells are transformed. Due to the low transformation efficiency, the survival of the transformed cells is difficult to assess. In contrast, the regeneration efficiency after bombardment is likely to be high due to the strongly dividing material that is treated. However, as TNE will occur in a single cell of a single callus, such an event will be easily lost if it is not selected for or, alternatively, regenerated plants will be chimeric for the targeted mutagenesis event. Thus, bombardment is not practical for performing targeted mutagenesis at non-selectable loci. In contrast, it is possible to recover targeted mutagenesis events using protoplasts as each microcallus is derived from a single protoplast.
Induced single nucleotide conversions by chimeras at ALS have demonstrated that targeted mutagenesis can be detected in tobacco, maize and rice cells. Bombardment has been used for tobacco (Beetham et al. 1999 Proc. Natl. Acad. Sci. USA 96: 8774-8778; Kochevenko et al. 2003 Plant Phys. 132: 174-184), maize (Zhu et al. 1999 Proc. Natl. Acad. Sci. USA 96, 8768-8773) and rice (Okuzaki et al. 2004 Plant Cell Rep. 22: 509-512). Beetham et al. (1999) reported that the frequency of herbicide resistance after direct transfer of chimeras increased fold compared to the background mutation rate (assumed to be 10−7 to 10−8). Kochevenko et al. have also used electroporation to perform targeted mutagenesis experiments in tobacco mesophyll protoplasts. The present inventors were able to obtain herbicide resistant tobacco callus at a frequency of 0.0001%, comparable to the frequency obtained by Beetham et al. This suggests that when dealing with the same plant species and the same target nucleotide that in this case the direct DNA delivery method does not have a large impact on the targeted mutagenesis efficiency, which remains at an undesirable low level. However, Ruiter et al. (2003 Plant Mol. Biol. 53, 715-729) performed both bombardment and electroporation experiments in both tobacco and oil seed rape, and could not detect any effect of the chimeras.
Bombardment of both maize and rice callus has been reported at an efficiency of 0.01% of the cells that are transformed (Zhu et al. (1999) Proc. Natl. Acad. Sci. USA 96, 8768-8773; Okuzaki et al. (2004(Plant Cell Rep. 22: 509-512). However, this is only feasible at selectable loci.
PEG-mediated protoplast transformation in itself has been known already since 1985. The first method for protoplast transformation utilized PEG (Krens et al. (1982) Nature 296: 72-74; Potyrykus et al. (1985) Plant Mol. Biol. Rep. 3:117-128; Negrutiu et al. (1987) Plant Mol. Biol. 8: 363-373). The technique is applicable to protoplasts from many different plants (Rasmussen et al. (1993) Plant Sci. 89: 199-207). PEG is thought to stimulate transformation by precipitating the DNA, in the presence of divalent cations, onto the surface of the protoplasts from where it then becomes internalized (Maas & Werr (1989) Plant Cell Rep. 8: 148-151). PEG transformation is the method of choice for transformation of Arabidopsis protoplasts (http://genetics.mgh.harvard.edu/sheenweb/faq.html) (Mathur et al. Methods in molecular biology, vol. 82, 267-276) and conforms well to the four requirements defined for a transformation method for efficient TNE. When tobacco protoplasts are treated with PEG, a biotin-labelled ss oligonucleotide can be detected in all cells examined. Survival, as assessed by vital staining using fluorescein diacetate, is >90% after PEG treatment. Not all protoplasts retain the ability to divide and form microcalli. In a typical isolation of non-treated tobacco protoplasts, approximately 25% form microcalli. PEG treatment does have a slight impact on regeneration efficiency, which drops to approximately 10%, but this is not dramatic compared to other transformation methods. None of the above describe prior art has contemplated the use of PEG transformation for site-directed mutagenesis, in particular TNE.
The present inventors have set out to improve the method of direct DNA transfer to obtain efficient targeted mutagenesis in plant cells. The present inventors have found that from amongst the transformation technologies as described herein elsewhere, PEG protoplast transformation enhances the overall targeted mutagenesis efficiency significantly compared to electroporation and biolistics. This is surprising, as the technologies for targeted mutagenesis in plants to date appeared to favour electroporation with the associated low efficiencies. Furthermore most improvements in the technology were directed at improving the mutagenic nucleobases and not in the delivery system for delivering the mutagenic nucleobase to the genomic target DNA.
For sake of comparison, the present inventors used ss mutagenic nucleobase designed to produce a P194Q conversion at the ALS locus leading to herbicide resistance. Identical ss mutagenic nucleobases were introduced into tobacco mesophyll protoplasts using either PEG mediated transformation or electroporation and herbicide resistant cells were selected using identical selection conditions. Thus the present inventors have found that PEG-mediated transformation of plant cells is the most efficient method to perform targeted mutagenesis in plant cells compared to known methods of transformation.
In one aspect the invention pertains to a method for targeted alteration of a duplex acceptor DNA sequence in a plant cell protoplast, comprising combining the duplex acceptor DNA sequence with a ss mutagenic nucleobase, wherein the duplex acceptor DNA sequence contains a first DNA sequence and a second DNA sequence which is the complement of the first DNA sequence and wherein the donor ss mutagenic nucleobase comprises at least one mismatch with respect to the duplex acceptor DNA sequence to be altered, preferably with respect to the first DNA sequence, wherein the method further comprises a step of introducing the ss mutagenic nucleobase into the cell protoplasts using polyethylene glycol (PEG) mediated transformation.
The ss mutagenic nucleobase is brought into contact with protoplasts of the plant to be transformed using a PEG transformation based technology. The PEG mediated transformation technology in itself is widely known and were necessary, small amendments to particular protocols can be made by the skilled man without departing from the gist of the present invention.
The ss mutagenic nucleobase used in the present invention have a length that is in line with other (chimeric) ss mutagenic nucleobase used in targeted mutagenesis, i.e. typically between 10-60 nucleotides, preferably 20-55 nucleotides, more preferably 25-50 nucleotides. In certain embodiments of the invention, the ss mutagenic nucleobase used in the present invention the can be modified, for instance by LNA and/or propynyl modifications as described in applicant's WO2007073166 and WO2007073170. Thus in certain embodiments, the ss mutagenic nucleobase contains at least one LNA located at a position that is from the targeted mismatch and preferably two LNAs located at least one nucleotide removed from either side of the mismatch and. Furthermore, these LNAs are at least 3, 4 or 5 nucleotides removed form the 5′ and/or 3′ ends of the ss mutagenic nucleobase. In certain embodiments, the ss mutagenic nucleobase can comprise one or more propyne substitutions, essentially as described in WO2007073166 and WO2007073170. In certain embodiments, the donor ss mutagenic nucleobase may be conjugated to protein such as a nuclear localisation signal. In this embodiment, the oligonucleotide used in the present invention is coupled via conventional (linker) technology to a nuclear localisation signal such as the known (NLS) peptide of the SV40 large T antigen, GATA transcription factor 11, DNA repair helicase XBP1, Light mediated protein DET1, ERF transcription factor, PR-related transcript activator PTI6 and nuclear coiled protein, essentially as described in applicants co-pending application PCT/NL2007/000279. The oligonucleotide-nuclear localisation signal conjugate can be used in the PEG-based transformation methodology described herein.
The alteration produced by the method of the present invention is a deletion, a substitution or an insertion of at least one nucleotide. Preferably the alteration is a substitution. More nucleotides may be altered in one oligonucleotide, but it is expected that efficiency will diminish, hence there is a preference for altering one nucleotide.
The target DNA (or duplex acceptor DNA) can be from any source, but preferably the target DNA is from a plant. Preferably the target DNA is from genomic DNA, linear DNA, artificial chromosomes, nuclear chromosomal DNA, organelle chromosomal DNA, episomal DNA. The method according to the invention can be used for altering a cell, correcting a mutation by restoration to wild type, inducing a mutation, inactivating an enzyme by disruption of coding region, modifying bioactivity of an enzyme by altering coding region, modifying a protein by disrupting the coding region.
In one aspect the invention relates to the use of PEG mediated transformation for enhancing the efficiency of targeted mutagenesis in plant protoplasts.
Without being bound by theory, it is thought that the use of PEG mediated transformation precipitates the DNA on the cell membrane of the protoplast. The precipitated DNA is encapsulated by the cell membrane and introduced into the protoplast in a shielded form.
The protoplast will, in the course of its normal cell cycle, directly after its formation by removal of the cell wall, start its normal cell wall regeneration process. The cell division typically starts later (from several hours up to a few days). The targeted nucleotide exchange generally takes place during the cell division, using the cell's repair mechanism. In the time period between the introduction of the donor DNA in the protoplast and the start of the cell division, the donor DNA is prone to attack form the cells defence mechanism such as exonucleases and is likely to be degenerated and hence become ineffective for TNE. With the use of PEG-mediated transformation technology, the donor DNA is encapsulated via endocytosis and is in this way at least temporarily shielded from the degenerative action of endonucleases. When the DNA is released from its encapsulated form, it has an increased chance of being present at or around the moment of the cell division, during which the DNA (i.e. the ss mutagenic nucleobase) is available to find its complement in the DNA of the acceptor cell and exchange the nucleotide as in common targeted mutagenesis mechanisms.